DESCRIPTION (Adapted from abstract): Exocytosis is a universal mechanism used by eukaryotic cells to export secretory products. It involves fusion of secretory vesicles with the plasma membrane by a hierarchical series of protein interactions and restructuring of the interaction phospholipid bilayers. Polyphosphoinositides appear to be regulators of exocytosis and are thought to act by binding proteins that control the fusion process. The long term objectives of this proposal are to characterize the interactions between polyphosphoinositides and integral membrane proteins of the secretory vesicles known as SCAMPs and to determine how these interactions contribute to exocytosis. Preliminary studies have shown that a unique peptide corresponding to a highly conserved segment within the primary structures of SCAMP family members inhibits exocytosis in rat peritoneal mast cells. The peptide also binds avidly to phospholipid vesicles containing phosphatidyl inositol-4,5-bisphosphate and to inositol-1,4,5-trisphosphate. Further studies are planned to identify the structural elements required for binding and to compare the binding affinities among related polyphosphoinositols and inositides and then correlate these results with potency in inhibiting exocytosis in mast cell. The aims of this study are to 1) analyze the molecular structure of the peptide/inositol phosphate complexes by NMR spectroscopy, 2) determine how the peptide binds membranes using site-specific spin labeling and EPR spectroscopy, 3) design mutant SCAMPs that will be expressed and tested as inhibitors of exocytosis and 4) use purified SCAMPs reconstituted in polyphosphoinositide-containing vesicles to confirm the binding of inositol lipid to full-length polypeptide and to test the prediction that binding causes a structural change that may reflect a role in catalysis of membrane fusion.